Process for removing immune complex in blood by use of the immobilized pepsin

ABSTRACT

The present invention relates to immobilized pepsin for use in removing immune complex which comprises pepsin fixed on a suitable carrier, and to a process for removing immune complex which comprises bringing blood or plasma from a patient suffering from an immune complex disease, such as rheumatoid arthritis and systemic erythematosus, into contact with the immobilized pepsin.

TECHNICAL FIELD

The present invention relates to immobilized pepsin for use in removingimmune complex from blood and to a process for removing immune complexfrom blood by the use of the immobilized pepsin.

BACKGROUND ART

Auto immune disorders or immune complex diseases, typified by rheumatoidarthritis, systemic lupus erythematosis (SLE), and lupus nephritis, are,as the names imply, disorders caused by a complex of various antigensand antibodies, that is, an immune complex. The mechanisms of immunecomplex diseases are so complicated that many points still remain forclarification; however, the diseses are considered generally to proceedas follows:

When tissues are damaged by bacterial or viaral infection, antibodiesare produced against newly formed autoantigens or virally infected cellsand the antibodies react with the corresponding antigens to form immunecomplexes. Since these immune complexes activate the complement systemand platelets, vasoactive substances such as histamine and serotonin arereleased and the permeability of the blood vessels is increased. Then,the immune complexes in circulation enter the vessel wall whosepermeabiity has been increased and deposit along the basement membrane.Polymorphonuclear leukocytes gather at the immune complex-deposited sitedue to the leukocyte chemotactic factors which have been formed by thereaction of the complement to the deposited immune complexes. Thepolymorphonuclear leukocytes, reacting with the immune complexes,release various tissue-damaging substances such as cathepsins D and E,collagenase, elastase and permeability factors, and these substanceseventually damage the tissue. In patients with immune complex diseasessuch as SLE, levels of the complement in the serum are generally low andaggravation of the disease conditions is closely correlated with thedecrease of the complement levels. This decline is thought to be due toplentiful consumption of the complement at the site of the reactionbetween antigens and antibodies taking place such as in the kidneys andblood vessels. Further, the immune complexes also are related to bloodcoagulation systems, and it is believed that the immune complexes causeserious symptoms through diverse mechanisms, for example by accelerationof fibrinoid deposition on the damaged tissues.

Currently in use for the treatment of these immune complex diseases arephysiotherapy and plasma-replacement therapy in addition to medicaltreatment with steroids, immunosuppressive agents, anti-inflammatoryagents and so forth. In particular, plasma-replacement therapy is one ofthe most reliable treating methods for removing immune complex, apathogenic factor, but the advantages of this method are not beingsufficiently utilized because of the difficulty in securing the supplyof plasma necessary for use in the plasma-replacement.

DESCRIPTION OF THE INVENTION

The present inventors carried out many intensive investigations todevelop a treating method which has the same efficacy asplasma-replacement therapy and yet does not require plasma forreplacement. As a result, they have found that pepsin and a pepsin-likeenzyme contained in leukocytes specifically decompose immune complex ata neutral pH region without affecting normal plasma proteins. On thebasis of this finding, they completed an invention relating to atreating agent for immune complex diseases which contains this enzyme asan effective ingredient, see Japanese patent application No. 18429/1981;PCT/JP82/00037. The present invention is an improvement over thatprevious one, and provides a process for removing immune complex withoutdamaging the inherent functions of plasma proteins. This processcomprises treating the plasma from a patient suffering from an immunecomplex disease with immobilized pepsin. The invention also provides theimmobilized pepsin for use in this process.

Since the major objective of plasma-replacement therapy resides inremoving nondialyzable pathogenic substances from a patient's plasma,especially immune complex, the treatment of a patient's plasma with theforegoing immobilized pepsin can have the same efficacy asplasma-replacement without the need to replace the plasma. For example,by subjecting plasma from a patient with an immune complex disease toextracorporeal circulation by passing the plasma through a column packedwith the above-mentioned immobilized pepsin, only immune complex isremoved from the plasma without affecting other plasma proteins.Moreover, since the present process does not require the use ofsubstitute plasma (from the patient or another person), the appearanceof side effects such as serum hepatitis or an adverse reaction due toincompatibility of blood can be left out of consideration.

The pepsin used in the present invention is a known enzyme (Journal ofClinical Investigations, Vol. 27, p. 818, 1948); the immobilized pepsinof the present invention can be obtained by purifying pepsinogencontained in the stomach, blood, urine, etc., of various animals by asuitable method, and fixing the purified pepsinogen on a suitablecarrier, followed by activating the immobilized pepsinogen in an acidtreatment. As the pepsin used for preparation of the immobilized pepsinof the present invention, pepsin derived from humans is preferablebecause it is the safest and the most effective, but pepsin derived fromanimals other than humans may also be used. As the carrier to be usedfor immobilization, carriers having less protein-adsorption capacitysuch as Sepharose® (a well-known adsorbent manufactured by PharmaciaFine Chemicals Co.), agarose, glass beads, etc., are preferable. It iseven more preferable to use pepsin immobilized on the inside surface ofthe filter membrane of an artificial dialyzer, or alternatively, a tubesuch as a nylon tube is used as a carrier to which pepsin can directlybe fixed. Known techniques can be utilized for the immobilization. Therecan also be employed an immobilized enzyme comprising a pepsin-likeenzyme fixed on a suitable carrier, wherein the purified pepsin-likeenzyme is obtained by passing a supernatant liquid of homogenizedleukocytes of a mammal through a DEAE-cellulose column equilibrated with0.1M acetate buffer (pH 5.3) to cause the pepsin-like enzyme to beadsorbed on the column, eluting the enzyme with the same buffercontaining 0.5M sodium chloride, and subjecting the eluate to gelchromatography on Sephadex G-100® (a well-known adsorbent manufacturedby Pharmacia Fine Chemicals Co.) swelled in 0.9% physiological saline.This pepsin-like enzyme has the following properties: (a) molecularweight of about 35,000 to 41,000; (b) isoelectric point of pH 2.5 to3.5; (c) maximum absorption at 278 nm; (d) positive ninhydrin reaction;(e) readily soluble in water and insoluble in ether and chloroform; and(f) white powdery appearance.

For example, according to the method of Seijffers et al (AmericanJournal of Physiology, Vol. 206, p. 1106, 1964), the immobilized enzymecan be prepared by passing human urine through a DEAE-cellulose columnequilibrated with 0.1M acetate buffer (pH 5.3) to cause pepsinogen to beadsorbed on the column, then eluting the pepsinogen with the same buffercontaining 0.3M sodium chloride, concentrating the eluate, furtherpurifying the concentrate by means of gel chromatography using SephadexG-100® swelled in 0.9% physiological saline, and coupling the purifiedproduct to Sepharose® by the use of cyanogen bromide to obtainimmobilized pepsinogen, followed by activating the pepsinogen underacidic conditions.

In carrying out the process of the present invention, it is convenientto utilize what is called extracorporeal circulation, wherein the bloodfrom a patient suffering an immune complex disease is guided to anextracorporeal circuit, and either the whole blood is treated with theforegoing immobilized pepsin, or only the plasma separated from theblood by a plasma separator is treated with the foregoing immobilizedpepsin and then recombined with the separated blood cell components, theblood thus treated being returned into the body. The treatment with theimmobilized pepsin is preferably carried out either by perfusing theplasma separated by a plasma separator through a column packed withpepsin which has been fixed on Sepharose particles or the like or byperfusing blood or plasma through a hollow tube which carriesimmobilized pepsin on its inner surface; however, the treatment is notlimited to these methods.

For the extracorporeal circulation of blood, it is convenient to use anapparatus composed of a pump for sending out the blood, a plasmaseparator for the separation of plasma from blood cells, and acontroller for the control of these devices. As such an apparatus, therecan be utilized the artificial liver-aiding apparatus of a separatedplasma perfusion type (Noboru Inoue: Chiryogaku (Therapeutics), Vol. 5,p. 477, 1980). In this method, generally 1-100 mg, preferably 5-50 mg,of the immobilized pepsin is suitably employed for 100 ml of blood, butthe amount of the immobilized pepsin can suitably be varied according tothe level of the immune complex. The present invention will next beexplained in more detail with reference to examples.

BEST MODE FOR CARRYING OUT THE INVENTION EXAMPLE 1

Distilled water was added to 150 ml of Sepharose 4B® (a well-knownadsorbent manufactured by Pharmacia Fine Chemicals Co.) up to 300 ml ofthe mixture, and the resulting mixture as adjusted to pH 11.0 with 6Nsodium hydroxide solution. To the mixture was then added 300 ml of 2.5%cyanogen bromide, and the pH of the mixture was maintained between 11.0and 11.5 for 30 minutes while the temperature was maintained at 16° C.The Sepharose® was washed with distilled water and with 0.1M sodiumbicarbonate solution, both of which had previously been cooled to 5° C.to remove the cyanogen bromide. The Sepharose® was then suspended in 300ml of the same solution. To 300 ml of the suspension was added 100 mg ofhuman urinary pepsinogen, and the mixture was reacted with gentlestirring at 5° C. for 16 hours to prepare immobilized pepsinogen. Theimmobilized pepsinogen was then activated at pH 2.0 for 10 minutes to beconverted into immobilized pepsin (pepsin 1 mg/ml resin), 100 ml ofwhich was packed into a glass column (4×8 cm).

Heparinized plasma (100 ml) from patients with rheumatoid arthritis andpatients with systemic lupus erythematosus who had been proved to carryimmune complex in their blood was passed through the immobilizedpepsin-column previously warmed to a temperature of 37° C. at the rateof 30 ml per hour. Taking human IgG aggregates as a standard substance,the amounts of immune complex in the plasma before and after thetreatment were determined by a hemolytic reaction of sheep erythrocytesusing guinea pig complements according to the method of Fust et al(Atherosclerosis, Vol. 29, p. 181, 1978); on the other hand, the amountsof albumin and γ-globulin in the plasma before and after the treatmentwere determined by electrophoresis using a cellulose acetate membrane(Ikagaku-jikken Koza (Lectures on Experiments in Medical Chemistry),Vol. 5, p. 201, 1973). Tables 1 and 2 show the results, respectively.

After passing the plasma through the immobilized pepsin column, thelevel of immune complex in the plasma decreased in the case of bothdiseases, whereas the normal plasma proteins underwent no changes.

                  TABLE 1                                                         ______________________________________                                                       Immune complex level                                                          (μg/ml)                                                                  No. of  Before     After                                         Disease      plasma  treatment  treatment                                     ______________________________________                                        Rheumatoid   1       184              63                                      arthritis    2       125        below 50                                                   3       134        below 50                                      Systemic lupus                                                                             1       403              123                                     erythematosus                                                                              2       253              71                                                   3       196        below 50                                      ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                                   Albumin     γ-Globulin                                                  (g/dl)      (g/dl)                                                          No. of  Before   After  Before After                                 Disease  plasma  treatment                                                                              treatment                                                                            treatment                                                                            treatment                             ______________________________________                                        Rheumatoid                                                                             1       4.3      4.3    0.8    0.8                                   arthritis                                                                              2       4.9      4.9    1.0    1.0                                            3       5.0      5.0    1.2    1.2                                   Systemic 1       4.8      4.8    1.3    1.3                                   lupus ery-                                                                             2       5.0      5.0    1.3    1.3                                   thematosus                                                                             3       5.1      5.1    1.2    1.2                                   ______________________________________                                    

EXAMPLE 2

In 10 ml of water was suspended 500 mg of aminopropylated glass beads,and the suspension was adjusted to pH 10 with an aqueous solution of 5Nsodium hydroxide. To the suspension was added 15 ml of tetrahydrofuransolution containing 1.5 g of cyanogen bromide, and the mixture was keptat pH 10 over a period of 10 minutes while adding an aqueous solution of5N sodium hydroxide at suitable intervals. The glass beads werecollected by filtration and washed with 0.1M sodium bicarbonatesolution. The glass beads thus treated were suspended in a mixed solventof 25 ml of 0.1M aqueous solution of sodium bicarbonate and 25 ml ofmethanol, which mixed solvent contained 3 moles of methyl11-aminoundecanoate hydrochloride, and the suspension was reacted at 4°C. overnight, while the pH was kept at 9. The glass beads were collectedby filtration, washed with water and methanol, and suspended in 20 ml ofabsolute methanol. To the suspension was added 1 ml of hydrazinehydrate, and the mixture was stirred for 3 hours. The glass beads werewashed with methanol, 0.1N hydrochloric acid, and water, in that order.Then, the beads were suspended in 20 ml of 1N hydrochloric acid. To thesuspension was added 280 mg of sodium nitrite, and the mixture wasreacted with stirring at 0° C. for 20 minutes.

After the completion of the reaction, the glass beads, washed with asmall amount of water, were suspended in 20 ml of 0.1M aqueous solutionof sodium bicarbonate (pH 8). Swine pepsinogen (50 mg) was added anddissolved into the suspension, and the mixture was reacted with stirringat 4° C. overnight. The immobilized pepsinogen thus obtained wasactivated at pH 2 by warming for 10 minutes to convert it intoimmobilized pepsin, which was suspended in 10 ml of 0.15M physiologicalsaline and packed into a glass column (1×8 cm). Heparinized plasma (10ml) containing immune complex (from patients suffering from rheumatoidarthritis and systemic lupus erythematosis, respectively) was passedthrough the foregoing column at the rate of 3 ml per hour. The immunecomplex level in the plasma and characteristics of other plasma proteinsbefore and after the treatment were examined in the same manner as inExample 1. Tables 3 and 4 show the results, respectively.

After passing the plasma through the immobilized pepsin column, theimmune complex level in the plasma decreased in the case of bothdiseases, whereas the normal plasma proteins underwent no changes.

                  TABLE 3                                                         ______________________________________                                                       Immune complex level                                                          (μg/ml)                                                                  No. of  Before     After                                         Disease      plasma  treatment  treatment                                     ______________________________________                                        Rheumatoid   1       193              58                                      arthritis    2       167              65                                                   3       143        below 50                                      Systemic lupus                                                                             1       353              134                                     erythematosis                                                                              2       296              88                                                   3       199        below 50                                      ______________________________________                                    

                  TABLE 4                                                         ______________________________________                                                   Albumin     γ-Globulin                                                  (g/dl)      (g/dl)                                                          No. of  Before   After  Before After                                 Disease  plasma  treatment                                                                              treatment                                                                            treatment                                                                            treatment                             ______________________________________                                        Rheumatoid                                                                             1       4.8      4.8    0.9    0.9                                   arthritis                                                                              2       4.7      4.7    1.0    1.0                                            3       5.0      5.0    1.0    1.0                                   Systemic 1       4.8      4.8    1.2    1.2                                   lupus ery-                                                                             2       5.1      5.1    1.2    1.2                                   thematosus                                                                             3       5.0      5.0    1.2    1.2                                   ______________________________________                                    

EXAMPLE 3

A nylon tube of 15 cm in length (inside diameter: 3 mm) was immersed ina water bath controlled at 35° C. Then the inside space of the tube wasfilled with 4.5N hydrochloric acid, and the tube was subjected toreaction for 15 minutes. The inside of the tube was washed withdistilled water and with 0.2M sodium bicarbonate solution (pH 9.4). Theinside space of the tube was then filled with 5% glutaraldehyde solutiondissolved in the foregoing solution, and the tube was subjected toreaction for 20 minutes. The inside surface of the tube was then washedsufficiently with 0.05M phosphate buffer (pH 8.0), and 5 mg of raturopepsinogen dissolved in the phosphate buffer was circulated throughthe tube for reaction at the rate of 0.2 ml/min for 2 hours so as to fixthe pepsinogen on the inside surface of the tube. The inside surface ofthe tube was then activated by treating with hydrochloric acid solutionof pH 2 for 10 minutes to convert the pepsinogen into immobilizedpepsin.

According to the method of Suzuki et al (Folia Pharmacologica Japonica,Vol. 68, p. 572 (1972)), anti-rat kidney rabbit serum was injectedintravenously at a dose of 5 ml/kg into Wistar strain male rats ingroups each consisting of 6 rats to induce nephritis. Twenty-one daysafter inducing the nephritis, 2 injection needles that had been treatedwith heparin were inserted and fixed in the jugular vein of each rat toform an extracorporeal circuit by connecting the 2 injection needleswith the foregoing tube, and the circulation was carried out at the rateof 2 ml per hour for 4 hours. As a control, a tube having no immobilizedpepsin was used in the same manner. The immune complex levels in theblood after the extracorporeal circulation were determined in the samemanner as in Example 1. Table 5 shows the results.

By the circulation of blood through the tube having an inside surfaceupon which was fixed immobilized pepsin, the immune complex level in theblood decreased remarkably.

                  TABLE 5                                                         ______________________________________                                                       Immune complex level                                                          (μg/ml)                                                     ______________________________________                                        Control group    283 ± 29                                                  Immobilized pepsin group                                                                        195 ± 11*                                                ______________________________________                                         *p < 0.05                                                                

EXAMPLE 4

A plasma separator of a filtration type with hollow threads of celluloseacetate was fitted into the cubital vein of each of 5 adult dogsanesthetized with pentobarbital (body weight: 10-15 kg). The immobilizedpepsin column prepared in Example 1 was connected to a heparinzedperfusion circuit for plasma, and the plasma was introduced to thecircuit at a controlled flow rate of 30 ml per hour by means of a flowrate adjustable pump (Perista pump Ato). The plasma which had passedthrough the immobilized pepsin column and the blood cell componentswhich had been separated were combined together, and returned into thebody through an extracorporeal circulation circuit formed with thecubital vein on the other side. 40 mg/kg of soluble immune complex(prepared at a ratio of human IgG: anti-human IgG rabbit antibody=4:1)was injected intravenously into the femoral vein by means of acontinuous infusion syringe (Truth A-II type) over a period of 3 hours.As a control, a Sepharose® column free of immobilized pepsin was used inthe same manner. Three hours after the extracorporeal circulation hadbeen started, the immune complex level in the plasma was determined inthe same manner as in Example 1. Table 6 shows the results.

After passing the plasma through the immobilized pepsin column, theimmune complex level in the plasma decreased remarkably.

                  TABLE 6                                                         ______________________________________                                                       Immune complex level                                                          (μg/ml)                                                     ______________________________________                                        Control group    343 ± 27                                                  Immobilized pepsin group                                                                        105 ± 11**                                               ______________________________________                                         **p < 0.01                                                               

EXAMPLE 5

100 mg of human gastric pepsinogen was fixed on the inside surface of afilter membrane of a hollow fiber type artificial dialyzer (Dow-4Cordis) with the use of cyanogen bromide and then activated withhydrochloric acid of pH 2.0 for 10 minutes to be converted intoimmobilized pepsin. Two hundred milliliters (5 cases) of heparinizedblood containing immune complex was circulated through the artificialdialyzer at the rate of 10 ml per minute. As a control, an artificialdialyzer having no immobilized pepsin was used in the same manner. After2 hours of circulation, the plasma was separated, and the immune complexlevel was determined according to the method of Example 1. Table 7 showsthe results.

After circulation of the blood through the immobilized pepsin, theimmune complex level in the blood decreased remarkably.

                  TABLE 7                                                         ______________________________________                                                       Immune complex level                                                          (μg/ml)                                                     ______________________________________                                        Control group    203 ± 19                                                  Immobilized pepsin group                                                                        128 ± 17*                                                ______________________________________                                         *p < 0.05                                                                

As described in the foregoing examples, the immobilized pepsin of thepresent invention decreases the amount of an immune complex in bloodwithout affecting in any way normal plasma proteins. Therefore, immunecomplex in the blood of a patient with an immune complex disease can beremoved without damage to physiological functions of the blood bytreating the blood or the plasma of the patient with the immobilizedpepsin, followed by returning the treated blood or plasma throughperfusion to the patient's body. This fact is extremely significantbecause it means that the utilization of the immobilized pepsin and theprocess for removing immune complex can produce the same result as isobtained by plasma replacement therapy, without a need for plasma forreplacement.

What is claimed is:
 1. A process for removing immune complexes fromblood or plasma, comprising contacting blood or plasma containing immunecomplexes with a material comprising an effective agent immobilized on apharmaceutically acceptable carrier, said agent being selected from thegroup consisting of pepsin and an enzyme derived from mammalianleukocytes, and recovering said blood or plasma freed of said immunecomplexes, said enzyme having the following properties:(a) molecularweight of about 35,000 to 41,000; (b) isoelectric point of pH 2.5 to3.5; (c) maximum absorption at 278 nm; (d) positive ninhydrin reaction;(e) readily soluble in water and insoluble in ether and chloroform; and(f) white powdery appearance.
 2. A process according to claim 1, whereinsaid agent is pepsin derived from human urine.
 3. A process according toclaim 1, wherein said agent is pepsin derived from a non-human animal.4. A process according to claim 1, wherein said carrier is selected fromthe group consisting of Sepharose, agarose and glass beads, and saidmaterial is packed into a column before being contacted with said bloodor plasma.
 5. A process according to claim 2, wherein said carrier isselected from the group consisting of Sepharose, agarose and glassbeads, and said material is packed into a column before being contactedwith said blood or plasma.
 6. A process according to claim 1, whereinsaid carrier includes a filter membrane of an artificial dialyzer.
 7. Aprocess according to claim 2, wherein said carrier includes a filtermembrane of an artificial dialyzer.
 8. A process according to claim 1,wherein said carrier includes an inside surface of a tube.
 9. A processaccording to claim 2, wherein said carrier includes an inside surface ofa tube.
 10. A method for treating a patient suffering from an immunecomplex disease, comprising contacting blood or plasma of said patientwith a material comprising an effective agent immobilized on apharmaceutically effective carrier, said agent being selected from thegroup consisting of pepsin and an enzyme derived from mammalianleukocytes, recovering said blood or plasma freed of said immunecomplexes, and returning said blood or plasma to said patient, saidenzyme having the following properties:(a) molecular weight of about35,000 to 41,000; (b) isoelectric point of pH 2.5 to 3.5; (c) maximumabsorption at 278 nm; (d) positive ninhydrin reaction; (e) readilysoluble in water and insoluble in ether and chloroform; and (f) whitepowdery appearance.
 11. A method according to claim 10, wherein saiddisease is selected from the group consisting of rheumatoid arthritis,systemic lupus erythematosis and lupus nephritis.
 12. A method accordingto claim 10, wherein said blood or plasma is contacted with saidmaterial in an extracorporeal circuit.
 13. A method according to claim10, wherein whole untreated blood is contacted with said material.
 14. Amethod according to claim 12, wherein said extracorporeal circuitincludes an artificial dialyzer, and said carrier includes a filtermembrane of said artificial dialyzer.
 15. A method according to claim10, wherein said carrier is selected from the group consisting ofSepharose, agarose and glass beads.
 16. A method according to claim 10,wherein said carrier includes an inside surface of a tube.
 17. A methodaccording to claim 10, wherein 1-100 mg of said material is contactedwith each 100 ml of blood or plasma.
 18. A method according to claim 10,wherein 5-50 mg of said material is contacted with 100 ml of said bloodor plasma.
 19. A method according to claim 10, wherein said blood orplasma is contacted with said material at a rate of 3-30 ml/hr.
 20. Amethod according to claim 10, wherein said agent is pepsin derived fromhuman urine.